Varicella zoster virus (VZV) is enveloped twice during its maturation in infected cells. Nucleocapsids first acquire a temporary envelope from the inner nuclear membrane as they bud into the perinuclear cisterna. This envelope enables the immature particles, which lack tegument, to fuse with the membrane of the rough endoplasmic reticulum (RER), delivering nucleocapsids to the cytosol. Viral glycoproteins (gps) and tegument come together at the trans-Golgi network (TGN), where the nucleocapsids receive their final envelope. We have found that VZV gpI is retrieved from the plasma membrane and targeted to the TGN because of a signal sequence (AYRV) and patch in its cytosolic domain (gpItail). Tegument, which is synthesized in the cytosol, adheres to the cytosolic face of a gpI-rich TGN-derived membrane that wraps nucleocapsids and becomes the viral envelope. We now propose to test the hypotheses that gps contain a TGN targeting signal of their own (which we will identify), or are routed to the TGN as passengers in a complex with a signal-containing navigator gp, such as gpI. Targeting and the formation of complexes will be studied in cells transfected or co-transfected with cDNA encoding the gps. We will also test the hypothesis that tegument proteins adhere to the TGN-derived membrane that envelops VZV because they bind to the cytosolic domains of one or more gps. The presence of a targeting signal in gpI tail implies that cells must contain proteins that interact with this signal. These are likely to be endogenous proteins involved in the traffic of vesicles between cytoplasmic compartments. Two methods will be used to identify cellular proteins that bind to the cytosolic domain of gpI: affinity chromatography with recombinant gpItails and a yeast-based 2 hybrid assay that detects the ability of two proteins to bind to one another by bringing a transcription activation domain into close proximity with a DNA-binding site that regulates the expression of a downstream reportergene. Cellular proteins will be eluted from gpItail with a synthetic peptide containing the gpI TGN targeting sequence, AYRV. A control peptide will be used to remove proteins that bind non-specifically to gpItail. For the yeast assay, we have constructed vectors containing hybrid genes that encode gpItail fused to the GAL4 binding domain. A cDNA library has been obtained in a corresponding vector encoding human brain proteins fused to the GAL4 activation domain. A third aim will be to determine whether the mannose 6-phosphate (Man 6-P) residues, which are present on viral gps, interact with the Man 6-P receptors (MPRs) that are present in the membranes of TGN-derived transport vesicles and may influence their post-TGN itinerary. Finally, we will investigate the signals that enable VZV to infect post-mitotic human neurons (hNT cells), a clinically important, but not well understood target of VZV. Specifically, we will test: (i) the role of MPRs at axon terminals in viral entry and (ii) the participation of a newly-discovered retrograde transport/nuclear import pathway in the translocation of immediate-early tegument proteins that contain a nuclear localization signal (such as IE62) from the cytosol of an axon terminal to the neuronal nucleus.